U.S. patent number 5,576,288 [Application Number 08/257,958] was granted by the patent office on 1996-11-19 for fibroblast growth factor conjugates.
This patent grant is currently assigned to The Salk Institute For Biological Studies. Invention is credited to J. Andrew Baird, Douglas A. Lappi.
United States Patent |
5,576,288 |
Lappi , et al. |
November 19, 1996 |
**Please see images for:
( Certificate of Correction ) ** |
Fibroblast growth factor conjugates
Abstract
The invention provides a conjugate comprising FGF or other
polypeptide reactive with an FGF receptor, and a cytotoxic agent.
The cytotoxic agent can be a ribosome-inactivating protein (RIP),
such as saporin, although other cytotoxic agents can also be
advantageously used. The cytotoxic agent can be attached to FGF
through a chemical bond, or the composition can be prepared as a
chimera using techniques of recombinant DNA. The conjugate can be
used to treat FGF-mediated pathophysiological conditions by
specifically targeting cells having FGF receptors and inhibiting
proliferation of or causing death of such cells. Additionally, the
conjugate can be used to target cytotoxic agents into cells having
FGF receptors to inhibit the proliferation of such cells. The
conjugate can be purified on an immobilized-heparin column.
Inventors: |
Lappi; Douglas A. (Del Mar,
CA), Baird; J. Andrew (San Diego, CA) |
Assignee: |
The Salk Institute For Biological
Studies (La Jolla, CA)
|
Family
ID: |
26698757 |
Appl.
No.: |
08/257,958 |
Filed: |
June 10, 1994 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
24682 |
Mar 1, 1993 |
|
|
|
|
344109 |
Apr 27, 1989 |
5191067 |
|
|
|
Current U.S.
Class: |
514/9.1;
424/94.5; 514/13.3; 514/19.3; 514/21.2; 514/6.9; 530/350; 530/370;
530/399; 530/402; 530/409 |
Current CPC
Class: |
A61K
47/642 (20170801) |
Current International
Class: |
A61K
47/48 (20060101); A61K 038/18 (); A61K 038/45 ();
C07K 014/50 (); C07K 014/415 () |
Field of
Search: |
;530/350,370,399,402,409
;514/2,8 ;424/94.5 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0259904 |
|
Mar 1988 |
|
EP |
|
8503508 |
|
Aug 1985 |
|
WO |
|
Other References
Huang et al "Association of Bovine Brain-Derived Growth Factor
Receptor . . . " J. Biol. Chem. 21:9568-9571 (Jul. 1986). .
Imamura et al "Purification of Basic FGF Receptors from Rat Brain"
Biochem Biophys. Res. Comm. 155(2):583-590 (Sep. 1988). .
Baird et al., "Angiogenic factor in human ocular fluid," The
Lancet, Sep. 7, 1985, p. 563. .
Baird et al., "Fibroblast growth factors," Brit. Med. Bull.,
45(2):438-452 (1989). .
Barbieri et al., "Ribosome-inactivating proteins from plants:
Properties and possible uses," Cancer Surveys, 1(3):489-520 (1982).
.
Gospodarowicz, D., "Isolation and characterization of acidic and
basic fibroblast growth factor," Methods in Enzymology, 147:106-119
(1987). .
Montecucchi et al., "N-terminal sequence of some
ribosome-inactivating proteins," Int. J. Peptide Protein Res.,
33:263-267 (1989). .
Lappi et al., "Characterization of a saponaria officinalis seed
ribosome-inactivating protein: Immunoreactivity and sequence
homologies," Biochem. and Biophys. Res. Comm., 129(3):934-942
(1985). .
Stirpe et al., "Ribosome-inactivating proteins from the seeds of
Saponaria officinalis L. (soapwort), of Agostemma githago L. (corn
cockle) and of Asparagus officinalis L. (asparagus), and from the
latex of Hura crepitans L. (sandbox tree)", Biochem. J.,
216:617-625 (1983). .
Blakey, et al., "Comparison of the pharmacokinetics and hepatotoxic
effects of saporin and ricin A-chain immunotoxins on murine liver
parenchymal cells," Cancer Research, 48(24pt1):7072-7078 (1988).
.
Akiyama et al., Verapamil enhances the toxicity of conjugates of
epidermal growth factor with Pseudomonas exotoxin and
antitransterrin receptor with Pseudomonas exotoxin, J. Cel. Phys.
120:271-279 (1984). .
Bacha et al., Thyrotropin-releasing hormone-diptheria toxin-related
polypeptide conjugates, J. Biol. Chem. 258:1565-1570 (1983). .
Bacha et al., Organ-specific binding of a thyrotropin-releasing
hormone-diptheria toxin complex after intravenous administration to
rats, Endocrinology 113:1072-1076 (1983). .
Baird et al., Molecular characterization of fibroblast growth
factor: Distribution and biological activities in various tissues,
Recent Progress in Hormone Res. 42:143-205 (1986). .
Baird et al., Receptor-and heparin-binding domains of basic
fibroblast growth factor, P.N.A.S. 85:2324-2328 (1988). .
Bergamaschi et al., Killing of K562 cells with conjugates between
human transferrin and a ribosome-inactivating protein (SO-6),
British J. of Haematology 68:379-384 (1988). .
Biro et al., "Stimulation and inhibition of protein synthesis in
endothelial and smooth muscle cells by bFGF-saporin," New York
Acad. Sci. Abstracts, p. 16, Jan. 16-18, 1991. .
Bregni et al., Activity of a Monoclonal antibody-saporin-6
conjugate against B-lymphoma cells, J. National Cancer Institute,
80:511-517 (1988). .
Case et al., Chimeric cytotoxin IL2-Pe40 delays and mitigates
adjuvant-induced arthritis in rats, P.N.A.S., 86:287-291 (1989).
.
Chaudhary et al., Activity of a recombinant fusion protein between
transforming growth factor type alpha and Pseudomonas toxin,
P.N.A.S., 84:4538-4542 (1987). .
Chaudhary et al., Selective killing of HIV-infected cells by
recombinant human CD4-Pseudomonas exotoxin hybrid protein, Nature,
355:369-372 (1988). .
Cuevas et al., Basic fibroblast growth factor (FGF) promotes
cartilage repair in vivo, Biochem. and Biophys. Res. Comm.,
156:611-618 (1988). .
Esch et al., Primary structure of bovine brain acidic fibroblast
growth factor (FGF), Biochem. and Biophys. Res. Comm., 133;554-562
(1985). .
Esch et al., Primary structure of bovine pituitay basic fibroblasts
growth factor (FGF) and comparison with the amino-terminal sequence
of bovine brain acidic FGF, P.N.A.S. 82:6507-6511 (1985). .
FitzGerald et al., Adenovirus-induced release of epidermal growth
factor and Pseudomonas into the cytosol of KB cells during
receptor-mediated endocytosis, Cell, 32;607-617 (1983). .
Folkman et al., Angiogenic factors, Science, 235;442-447 (1987).
.
Grindey, "Current status of cancer drug development: Failure of
limited success?," Cancer Cells 2(6):163-171 (1990). .
Halaban et al., bFGF as an autocrine growth factor for human
melanomas, Oncogene Research, 3:177-186 (1988). .
Kelley et al., Interleukin 2-diphtheria toxin fusion protein can
abolish cell-mediated immunity in vivo, P.N.A.S., 85:3980-3984
(1988). .
Lappi et al., Biological and chemical characterization of basic
EGF-saporin mitotoxin, Biochem. Biophys. Res. Commun., 160:917-923
(1989). .
Lorberboum-Galski et al., Cardiac allograft survival in mice
treated with IL-2-PE40, P.N.A.S., 86:1008-1012 (1989). .
Lorberboum-Galski et al., Cytotoxic activity of an interleukin
2-Pseudomonal exotoxin cimeric protein produced in Escherichia
coli, P.N.A.S., 85:1922-1926 (Mar. 1988). .
Lorberboum-Galski et al., Interleukin 2 (IL2) PE40 is cytotoxic to
cells displaying either the p55 or p70 subunit of the IL2 receptor,
J. Biol. Chem. 263:18650-8656 (1988). .
Schwartz et al., A new cytotoxin specific for the target cells of
corticotropin-releasing factor, Endocrinology 121:1454-1460 (1987).
.
Siegall et al., FASEB Journal, 5:2843-2849 (1991). .
Siegall et al., Cytotoxic activities of a fusion comprised of
TGFalpha and Pseudomonas exotoxin, FASEB Journal 3:2647-2652
(1989). .
Siegall et al., Cytotoxic activity of an interleukin 2-Pseudomonas
exotoxin, P.N.A.S., 85:9738-9742 (1988). .
Siena et al., Evaluation of antihuman T lymphocyte saporin
immunotoxins potentially useful in human transplantation,
Transplantation, 46:747-753 (1988). .
Siena et al., Synthesis and characterization of an antihuman
T-lymphocyte saporin immunotoxin (OKT-1-SAP) with in vivo stability
into nonhuman primates, Blood, 72:756-765 (1988). .
Taetle et al., Effects of anti-epidermal growth factor (EGF)
receptor antibodies and an anti-EGF receptor recombinant-ricin A
chain immunoconjugate on growth of human cells, J. Nat. Cancer
Inst., 80(13):1053-1059 (1988). .
Walicke, et al., Neurotrophic effects of basic and acidic
fibroblast growth factors are not mediated through glial cells,
Developmental Brain Research, 40:71-79 (1988). .
Vollmar et al., Toxicity of ligand and antibody-directed ricin
A-chain conjugates recognizing the epidermal growth factor
receptor, J. Cell. Phys. 131:418-425 (1987)..
|
Primary Examiner: Walsh; Stephen G.
Attorney, Agent or Firm: Fitch, Even, Tabin &
Flannery
Government Interests
This invention was made with Government support under Grant
DK-18811 awarded by the National Institutes of Health (DHHS). The
Government has certain rights in this invention.
Parent Case Text
This is a continuation of application Ser. No. 08/024,682 filed on
Mar. 1, 1993, now abandoned, which is a continuation-in-part of
U.S. application Ser. No. 07/344,109, filed Apr. 27, 1989, now U.S.
Pat. No. 5,191,067.
Claims
We claim:
1. A conjugate, comprising a cytotoxic agent and a polypeptide
reactive with a high affinity fibroblast growth factor (FGF)
receptor, wherein the polypeptide reactive with the receptor is
selected from the group of polypeptides consisting of polypeptides
that exhibit FGF mitogenic activity mediated through binding to an
FGF receptor and fragments of polypeptides that exhibit FGF
mitogenic activity mediated through binding to an FGF receptor and
that bind to an FGF receptor and are transported into the cell,
thereby internalizing the linked cytotoxic agent.
2. The conjugate of claim 1, wherein the polypeptide reactive with
an FGF receptor is basic FGF or a fragment thereof.
3. The conjugate of claim 1, wherein the polypeptide reactive with
an FGF receptor is selected from the group consisting of acidic
FGF, the hst gene product, the in t-2 gene product and FGF-5, or a
fragment thereof.
4. The conjugate of claim 1 wherein said cytotoxic agent is a
ribosome-inactivating protein.
5. The conjugate of claim 4 wherein said cytotoxic agent is a
saporin.
6. The conjugate of claim 1, wherein the cytotoxic agent is
selected from the group consisting of methotrexate, anthracyclines
and Pseudomonas exotoxin.
7. A pharmaceutical composition, comprising the conjugate of claim
1 and a physiologically acceptable excipient.
8. The conjugate of claim 1, wherein the polypeptide reactive with
an FGF receptor is acidic FGF or a fragment thereof.
9. The conjugate of claim 1, wherein the FGF receptor is a receptor
to which basic FGF (bFGF) binds and which transports bFGF into the
cell.
Description
This application relates to compositions which inhibit cell
proliferation, and, more specifically, to fibroblast growth factor
conjugated to a cytotoxic agent.
BACKGROUND OF THE INVENTION
A great deal of attention has been directed towards the
identification and characterization of factors capable of
stimulating the growth and proliferation of specific cell types. In
the last twenty-five years, a number of such mitogenic factors have
been isolated. Rather than having highly specific activities as may
have been originally anticipated, many such growth factors are now
recognized to have multifunctional activities, affecting a wide
spectrum of cell types. In addition, certain activities are shared
by homologous members of a family of growth factors.
One family of growth factors now known to have a broad spectrum of
activities is the fibroblast growth factors (FGF). Basic FGF is a
protein which has a molecular weight of approximately 16 kD, is
acid and temperature sensitive and has a high isoelectric point. A
structurally related protein, acidic FGF, has an acidic isoelectric
point. FGFs exhibit a mitogenic effect on a wide variety of
mesenchymal, endocrine and neural cells. Of particular interest is
their stimulatory effect on collateral vascularization and
angiogenesis. Such mitogenic effects have stimulated considerable
interest in FGF as potential therapeutic agents for wound healing,
nerve regeneration and cartilage repair, for example.
Cells that respond to basic FGF have been shown to possess specific
receptors on the cell surface membranes. The receptor proteins
appear to be single chain polypeptides with molecular weights
ranging from 110 to 150 kD, depending on cell type. The proteins
bind basic FGF with high affinity (Kd=10-80 pM), with receptor
numbers ranging from 2000 to 80,000 per cell. The receptors can be
purified from rat brain, using a combination of lectin and ligand
affinity chromatography and are associated with tyrosine kinase
activity (Imamura et al., Biochem. Biophys. Res. Comm., 155:583-590
(1988); Huang and Huang, J. Biol. Chem., 261:9568-9571 (1986), both
of which are incorporated herein by reference).
On baby hamster kidney cells (BHK), two basic FGF receptors with
estimated molecular weights of 110 and 130 kD have been reported
(Neufeld and Gospodarowicz, J. Biol. Chem., 260;13860-13868 (1985);
Neufeld and Gospodarowicz, J. Biol. Chem., 261:5631-5637 (1986),
both of which are incorporated herein by reference). Both receptor
proteins bind basic FGF and acidic FGF, although it appears that
the larger binds basic FGF preferentially while the smaller has
somewhat higher affinity for acidic FGF.
In addition to potentially useful proliferative effects, basic
FGF-induced mitogenic stimulation may, in some instances, be
detrimental. For example, cell proliferation and angiogenesis are
an integral aspect of tumor growth. Basic FGF is thought to play a
pathophysiological role, for example, in tumor development,
rheumatoid arthritis, proliferative diabetic retinopathies and
other complications of diabetes.
There thus exists a need for being able to inhibit certain
mitogenic effects of basic FGF within the body which may give rise
to pathological conditions; however, such inhibition must be
accomplished in a way that does not result in the death of the
animal or the infliction of substantial harm thereto. Because of
the ubiquitous distribution of FGF target cells and presumably FGF
receptors throughout the body, it was felt that such an objective
could not be accomplished, yet the present invention satisfies this
need.
SUMMARY OF THE INVENTION
The invention provides a conjugate comprising basic FGF or other
polypeptide reactive with an FGF receptor, and a cytotoxic agent.
In one embodiment, the cytotoxic agent is a ribosome-inactivating
protein (RIP), such as, for example, saporin, although other
cytotoxic agents can also be advantageously used. The cytotoxic
agent can be attached to basic FGF through a chemical bond or the
composition can be prepared as a chimera, using techniques of
recombinant DNA. In both cases, the conjugate molecule is designed
and produced in such a way that the receptor-binding epitope of the
basic FGF moiety of the complex is left available for recognition
by the FGF receptor.
The conjugate can be used to treat FGF-mediated pathophysiological
conditions by specifically targeting to cells having FGF receptors
and inhibiting proliferation of or causing death of the cells. Such
pathophysiological conditions include, for example, tumor
development, Dupuytren's Contracture, certain complications of
diabetes such as proliferative diabetic retinopathies, and
rheumatoid arthritis. The treatment is effected by administering a
therapeutically effective amount of the FGF conjugate, for example,
in a physiologically acceptable excipient. Additionally, the
conjugate can be used to target cytotoxic agents into cells having
FGF receptors, and to inhibit the proliferation of such cells. A
method of purifying the conjugate on a heparin-immobilized column
is also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a heparin Sepharose chromatography of the conjugation
reaction mixture.
FIG. 2A and FIG. 2B show the RIP and binding activities of the
basic FGF/SAP conjugate. The activity was compared to SAP alone in
a cell-free protein synthesis inhibition assay (FIG. 2A) (SAP
.box-solid., basic FGF-SAP .oval-solid.) and the receptor binding
activity was compared to basic FGF in the BHK radioreceptor assay
(FIG. 2B) (basic FGF .quadrature., basic FGF-SAP .oval-solid.).
Each point is the mean of 3 replicates. Standard deviations were
less than 10%.
FIG. 3 shows the effect of basic FGF/SAP on BHK cell proliferation.
Cell counts were normalized to media controls (190,000.+-.15,000).
Cell number with 10.sup.-8 M of the mitotoxin was 9,527.+-.980. N=3
in all instances. (Basic FGF-SAP .oval-solid., SAP .box-solid.,
basic FGF .quadrature., basic FGF+SAP .largecircle.)
FIG. 4 shows the effect of exogenous basic FGF and NGF on
cytotoxicity. Basic FGF-SAP was used at a concentration of
10.sup.-10 M basic FGF-AP and C: preincubation with equimolar free
of basic FGF; D: 10-fold excess of free basic FGF; E: 100-fold
excess of basic FGF; F: 1000-fold excess of basic FGF; G: equimolar
incubation with equimolar free NGF; H: 10-fold molar excess; I:
100-fold molar excess, J: 100-fold molar excess.
FIG. 5 shows the relationship between toxicity of basic FGF-SAP and
FGF receptor number, determined for each cell line after 48 or 72
hours exposure to basic FGF-SAP. Cell numbers were determined and
the concentration that reduced the number of cells by 50% was
plotted against receptor number for that cell line. Receptor number
was determined by the method of Moscatelli et al., J. Cell
Physiol., 131:123-130 (1987).
FIG. 6 shows the effect of basic FGF-SAP on Dupuytren's Cells as
described in Example IV.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a conjugate comprising FGF, or
polypeptide fragments thereof, reactive with an FGF receptor and a
cytotoxic agent, which composition is effective for inhibiting
growth and proliferation of cells having FGF receptors. The
composition can be used to counteract the mitogenic effects of
basic FGF, where such an effect is deleterious, such as in tumor
angiogenesis and proliferative complications of diabetes, such as
proliferative retinopathies.
As used herein, the term "FGF" refers to both basic FGF (bFGF) and
acidic FGF (aFGF) and other proteins, or fragments thereof,
exhibiting FGF mitogenic activity mediated through binding to an
FGF receptor. For example, a basic FGF peptide having a molecular
weight of about 16 kD and a pI of about 9.6 has been described by
Esch et al. Other FGF proteins include other forms of basic FGF
which have an amino terminal extension, aFGF, hst oncogene, int-2
oncogene, FGF-5, and FGF-6. (See Baird et al., Brit. Med. Bull.,
45:438-452 (1989)). FGF fragments include FGF peptides of between
about 5-100, preferably about 5-50, more preferably about 5-25
amino acids that are able to bind to an FGF receptor. See Baird et
al., PNAS, 85:2324-2328 (1988), which is incorporated herein by
reference, for an example of basic FGF peptide fragments that are
reactive with an FGF receptor. Conjugates of acidic fibroblast
growth factor fused to several mutant forms of Pseudomonas exotoxin
have been shown to be cytotoxic to a variety of tumor cell lines
including hepatocellular, prostatic, colon, and breast carcinomas
(Siegall et al., The FASEB Journal, 2843-2849, October, 1991).
FGF expresses mitogenic activity in a wide variety of normal
diploid mesoderm-derived and neural crest-derived cells. A test of
such "FGF mitogenic activity" is the ability to stimulate
proliferation of cultured bovine aortic endothelial cells, as
described in Gospodarowicz et al., J. Biol. Chem., 257:12266-12278
(1982); Gospodarowicz et al., Proc. Natl. Acad. Sci. USA,
73:4120-4124 (1976), which are incorporated herein by
reference.
The term FGF is used to refer both to proteins having amino acid
sequences found in a mammalian host, as well as to modified
sequences, having amino acid substitutions, deletions, insertions
or additions, which still express mitogenic activity, mediated
through binding to an FGF receptor. Purified preparations of basic
FGF and acidic FGF are frequently observed to include several
molecular forms of the mitogens. It is understood that differences
in amino acid sequences can occur in FGF from different species as
well as between FGF from individual organisms of a particular
species. The term is intended to refer to both proteins isolated
from natural sources as well as those made synthetically, as by
chemical synthesis or recombinant means.
The amino acid sequence of an exemplary mammalian basic FGF derived
from bovine pituitary tissue is provided in Esch et al., Proc.
Natl. Acad. Sci. USA, 82:6507-6511 (1985), which is incorporated
herein by reference. As used herein, the term "basic FGF" refers to
proteins or polypeptides having substantially the same amino acid
sequence and mitogenic activity as that of the basic FGF described
in Esch, supra. cDNAs encoding human aFGF (Jaye et al., Science,
233:541-545 (1986)) and bovine (Abraham et al., Science,
233:545-548 (1986), human (Abraham et al., EMBO J., 5:2523-2528
(1986); Abraham et al., Quant. Biol., 51:657-668 (1986)), and rat
(Shimasaki et al., Biochem. Biophys. Res. Commun. (1988); Kurokawa
et al., Nucleic Acids Res., 16:5201 (1988)) basic FGF have been
cloned, and sequenced and predict the existence of proteins
identical to those found by protein sequencing.
As used herein, the term "FGF receptors" refers to receptors which
are able to bind basic FGF and transport it into the cell. Included
among these are the receptors described in Imamura, J. Cell
Physiol., 131:123-130 (1987) and Moscatelli, supra. As used herein,
the term "polypeptide reactive with the FGF receptor" refers to any
polypeptide which is capable of binding an FGF receptor and of
being transported into the cell thereby.
Basic FGF is commercially available, for example, from Amgen
(Thousand Oaks, Calif.). Acidic FGF is also commercially available
from Promega (Madison, Wis.) Basic and acidic FGF can be obtained
from a variety of tissue types of mammals. For example, methods of
purifying basic FGF using reverse-phase high performance liquid
chromatography (RP-HPLC), heparin-Sepharose affinity chromatography
and cation exchange HPLC and RP-HPLC are described in U.S. Pat. No.
4,785,079, as well as Gospodarowicz et al., Proc. Natl. Acad. Sci.,
81:6963-6967 (1984) and Gospodarowicz, Meth. Enzym., 147:106-119
(1987), which are incorporated herein by reference. In addition,
basic FGF can be synthesized, as by chemical or recombinant
methods. Expression of a recombinant protein in yeast and E. coli
is described in Barr et al., J. Biol. Chem., 263:16471-16478
(1988), which is incorporated herein by reference.
The FGF-cytotoxic agent conjugate can be purified on a column
containing immobilized heparin. Appropriate columns include
heparin-Sepharose and heparin-agarose. The bound conjugate can be
eluted with a gradient salt, such as NaCl and is eluted between 1
and 3M.
According to one aspect of the invention, basic FGF is conjugated
to a cytotoxic agent so as to target the cytotoxic agent
specifically to cells which exhibit FGF receptors. As used herein,
the term cytotoxic agent refers to a molecule capable of inhibiting
cell function. The term includes agents which are only toxic when
transported into the cell and also those whose toxic effect is
mediated at the cell surface. A variety of cytotoxic agents can be
used including those which inhibit protein synthesis.
In one aspect of the invention, FGF is combined with a
ribosome-inactivating protein (RIP) such as, for example, the
type-1 RIP saporin-6 (SAP) or other SAP derivatives. SAP is a
potent RIP which is isolated from the seeds of the plant Saponaria
officinalis (see Stirpe, et al., Biochem. J., 216:617-625 (1983)).
Other suitable RIPs include, but are not limited to, ricin, ricin A
chain, gelonin, diphtheria toxin, diphtheria toxin A chain,
trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis
antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin,
bryodin, and shiga. L. Barbieri et al., Cancer Surveys, 1, 489-520
(1982) and EPO published patent application No. 466,222,
incorporated herein by reference, provide lists of numerous RIPs
and their sources.
Other cytotoxic agents which are considered to be functionally
equivalent to the aforementioned RIPs include Pseudomonas exotoxin
and metabolic inhibitors which are known in this art, but are not
limited thereto. Therefore, the term RIPs is used in this
application to broadly include such cytotoxins. For example,
chimeric proteins composed of acidic fibroblast growth factor fused
to several mutant forms of Pseudomonas exotoxin have been produced
that have proven to be cytotoxic to a variety of tumor cell lines,
including hepatocellular, prostatic, colon, and breast carcinomas
(Siegall et al., The FASEB Journal, 5:2843-2849, October, 1991).
The pseudomonas toxin has also been shown to be effective at
killing cells expressing epidermal growth factor receptors when
fused to transforming growth factor type .alpha. as a chimeric
protein. Chaudhary et al., PNAS, 84:4538-4542, (1987).
In another aspect of the invention, the cytotoxic agent is a drug.
Examples of such drugs are anthracyclines such as the daunomycins
(including daunorubicin and doxorubicin) and methotrexate and its
analogs. Others are known to those skilled in the art.
FGF can be conjugated to a protein cytotoxic agent by any means
known to those skilled in the art, such as through derivitization
with a reactive sulfhydryl containing moiety such as SPDP, or via a
suitable cross linking agent, such as glutaraldehyde or
carbodiimide. In one embodiment, the cytotoxic agent is derivatized
with a reactive sulfhydryl containing agent, such as
N-succinimidyl-3(2-pyridyldithio)propionate. FGF is then added to
and mixed with the derivatized cytotoxic agent. The FGF conjugate
can be separated from the unreacted products on a column.
Alternatively, FGF can be conjugated to a drug, such as 14 bromo
doxorubicin through the sugar moiety, as by the cis-aconitate
method (Shen and Riser, BBRC, 102:1048 (1981), which is
incorporated herein by reference).
Alternatively, chimeric FGF-conjugates can be prepared by
recombinant methods. Such methods as applied to conjugates of IL-2
or TGF.alpha. are provided in Chaudhary et al., Proc. Natl. Acad.
Sci. USA., 84:4538-4542 (1987) and Lorberman-Galski et al., Proc.
Natl. Acad. Sci. USA, 85:1922-1926 (1988), which are incorporated
herein by reference. See also, Maniatis, et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory (1982), which is
incorporated herein by reference.
A conjugate containing FGF and a cytotoxic agent is useful in
treating a variety of FGF-mediated pathophysiological conditions.
As used herein, the term "FGF-mediated pathophysiological
condition" refers to a deleterious condition characterized by or
caused by proliferation of cells which are sensitive to basic FGF
mitogenic stimulation. Basic FGF-mediated pathophysiological
conditions include, but are not limited to, tumors, rheumatoid
arthritis, Dupuytren's Contracture and certain complications of
diabetes such as proliferative retinopathy.
FGF-cytotoxic agent conjugates can be used to target the cytotoxic
agent to cells expressing FGF receptors in order to cause cell
death. Surprisingly, there is a direct relationship between the
number of FGF receptors per cell and the dose at which 50% of the
cells are killed (the ED.sub.50), as is shown in FIG. 5. Moreover,
for cells with extremely high receptor numbers, for example, BHK
cells, the ED.sub.50 is identical to the affinity constant of basic
FGF for its receptor (both are about 25 pM for BHK cells). This
unexpected result indicates that the presence of the cytotoxic
agent, even such a large molecule as SAP, does not reduce basic FGF
activity. Moreover, these results indicate that these cells that
are expressing a large number of basic FGF receptors are
particularly sensitive to the conjugate.
In order to treat FGF-mediated pathophysiological conditions, a
therapeutically effective amount of FGF-cytotoxic agent conjugate
is administered to a mammal in a physiologically acceptable
excipient. Examples of physiologically acceptable excipients
include PBS and saline.
The following examples are intended to illustrate but not limit the
invention.
EXAMPLE I
Conjugation of FGF With Saporin
Recombinant basic FGF corresponding to the sequence of 154 amino
acids (Abraham et al., Quant. Biol., 51:657-668 (1986), which is
incorporated herein by reference) was obtained from Farmitalia
Carlo Erba. Saporin-6 was purified according to the method of
Stirpe, et al., supra, as modified by Lappi, et al., Biochem.
Biophys, Res. Comm,, 129:934-942 (1985), which is incorporated
herein by reference. Briefly, seeds of Saponaria officinalis were
extracted by grinding in 0.14M NaCl in 5 mM sodium phosphate
buffer, pH 7.2 (8 ml/g). After overnight stirring at 4.degree. C.,
extracts were strained through cheese-cloth and were centrifuged at
28000 g for 30 minutes. The supernatant was separated from the
sediment and from floating fat, and is referred to as "crude
extract."
Crude extracts were dialyzed against 5 mM sodium phosphate buffer,
pH 6.5, centrifuged at 28000 g for 30 minutes and applied to a CM
cellulose column (CM 52; Whatman, Maidstone, Kent, U.K.), which
after washing, was eluted with a 0-0.3M NaCl gradient in the same
buffer. This material was then dialyzed against water and
chromatographed on an FPLC Mono S column (Pharmacia, Uppsala,
Sweden) equilibrated with 50 mM sodium borate pH 9.5, 0.156M sodium
chloride. The protein was eluted with a 20 minute gradient from
0.156M to 0.186M sodium chloride. The resultant peak material was
then extensively dialyzed against Milli-Q water (Millipore,
Bedford, Mass.). A portion of the dried material was weighed and
dissolved in water at a concentration of 1 mg/ml. An ultraviolet
spectrum was recorded giving a 1% extinction coefficient of 6.4 at
277 nm, the absorbance maximum. At 280 nm the E.sub.280 was 6.0. A
protein assay using the Lowry method (Lowry, et al., J. Biol.
Chem., 193:265-275 (1951)) using BSA as a standard gave a result of
1.07 mg/ml.
SAP was derivatized with
N-succinimidyl-3(2-pyridyldithio)propionate (SPDP; Pharmacia Fine
Chemicals, Piscataway, N.J.) according to the manufacturer's
instructions. Briefly, SAP was dissolved in (2.7 mg/ML) sodium
phosphate buffer (0.1M, pH 7.5) containing NaCl (0.1M). A 1.25
molar excess of SPDP, dissolved in ethanol, was added drop by drop
while stirring, and allowed to react for 30 minutes at 23.degree.
C. with occasional stirring. Excess reagent and low molecular
weight reaction products were removed by gel filtration. Basic FGF
(2 mg/ml) was added to and mixed with the derivatized saporin (6
mg/ml in 0.1M sodium phosphate, 0.1M sodium chloride, pH 7.5) for
two hours at room temperature. The reaction was terminated by the
addition of 35 .mu.L of 0.1M iodacetamide. After an additional 30
minutes, the reaction mixture was diluted to 30 ml and loaded onto
a heparin-Sepharose (Pharmacia) column (0.5.times.5.5 cm). The
bound proteins were eluted with a step gradient of 0.6M, 1M and 2M
NaCl in 10 mM TRIS, pH 7.4. The material eluting between 1M and 2M
was pooled. Final purification of the conjugate was achieved after
the pool was dialyzed against water and chromatographed on a Mono S
5/5 NaCl cation exchange column (Pharmacia) (buffer A: 50 mM sodium
borate, pH 8.0, buffer B: 0.5M NaCl in buffer A). Fractions
containing the conjugate were detected by silver staining after
PhastGel (Pharmacia) electrophoresis and appropriate fractions were
pooled for analysis.
Synthesis of the conjugate was assessed by gel electrophoresis and
allowed to proceed until no detectable basic FGF remained in the
reaction mixture. Chromatography on heparin-Sepharose (FIG. 1) and
subsequent electrophoretic analysis of each of the peak fractions
showed that, while SAP does not bind to heparin-Sepharose, the
conjugate does. Only small amounts of the conjugate were released
during the 1.0M NaCl wash. The major product eluted with the 2M
wash and contained equimolar amounts of SAP and basic FGF
(Mr.about.40,000). However, there was also a portion of the
conjugate that has an estimated Mr>68,000 presumably as a result
of the conjugation of two molecules of basic FGF per molecule of
saporin.
Unambiguous identification of the SAP-basic FGF conjugate was
accomplished using sequence specific antisera raised in rabbits.
The immunogen used was a fragment of basic FGF comprising amino
acids 1 through 24, chemically synthesized using a 990 Peptide
Synthesizer (Beckman Instruments, Brea, Calif.). Western blot
analysis showed that all molecular weight forms of the conjugate
contained both basic FGF and SAP. The antiserum recognizes the
midportion of the peptide and cross-reacts on equimolar basis with
purified bovine and recombinant human basic FGF.
Samples in a sodium dodecyl sulfate-containing polyacrylamide gel,
after electrophoresis, were electroblotted onto nitrocellulose
membranes, and allowed to air dry. The membrane was covered with
TRIS-buffered saline (TBS) and agitated for 10 minutes. The
solution was aspirated and discarded. The membrane was covered with
5% nonfat milk (NFM) in TBS and agitated for 10 minutes. The
solution was aspirated and discarded. Primary antibody, either
anti-SAP or anti-basic FGF anti-serum, at a concentration of 1/100
in NFM/TBS was added and agitated overnight. The solution was
aspirated and discarded. The membrane was covered with TBS,
agitated for 10 minutes and the solution aspirated and discarded.
The membrane was covered with 0.05% NP40/TBS and shaken 1 minute;
the solution was aspirated and discarded. The final TBS and
NP40/TBS washes were replated twice. Horseradish peroxidase
labelled anti-IgG at a dilution of 1/2000 in NFM/TBS was added and
the membrane agitated for 2 hours. The TBS and NP40/TBS wash steps
were repeated. The membrane was placed in a solution (Freshly
mixed) of 60 mg 4-chloro-1-naphthol in 20 mL methanol and 100 mL
double distilled water and 10 .mu.L 30% H.sub.2 O and allowed to
develop. The solution was aspirated and discarded and the reaction
stopped by rinsing with water. The membrane was allowed to dry.
EXAMPLE II
Activity of the FGF-SAP Conjugate
The capacity of the conjugate to recognize the basic FGF receptor
was examined in BHK cells using the procedure described by
Moscatelli, et al., J. Cell Physiol., 131:123-130 (1987), which is
incorporated herein by reference. Briefly, cells were grown to
subconfluence and incubated in 300 .mu.L buffer containing F-12 14
mM NaHCO.sub.3, 25 mM HEPES and 0.2% gelatin at 4.degree. C. for
two hours with 10 .mu.L radioiodinated basic FGF in the presence of
various concentrations of basic FGF or the conjugate. The cells
were then washed three times with 0.5 mL phosphate-buffered saline
(PBS), and twice with 2M NaCl in PBS. Binding to the high affinity
receptor was determined by counting the membrane fraction that was
solubilized 0.5% Triton X-100 in PBS (pH 8.1).
The protein synthesis inhibition activity of the SAP protein was
compared to the protein synthesis inhibition activity of the basic
FGF-SAP conjugate in in vitro assays of protein synthesis as
described in Siehn et al., Blood, 72:756-765 (1988), which is
incorporated herein by reference. The cytotoxic activity of the
conjugate was tested on baby hamster kidney fibroblasts (ATCC
Accession No. CRL 6281). BHK cells were plated in 24 well plates at
a concentration of 5000 cells/ml and incubated overnight at
37.degree. C., 5% CO.sub.2. The following morning, HEPES-buffered
DMEM and F-12 media (1:1) plus 5% FCS was aspirated from the wells
and replaced with media alone or with media containing the
conjugate, basic FGF or saporin. Two days later, the cells were
washed twice, trypsinized and cell number determined with a Coulter
Particle Counter (Coulter Electronics, Hialeah, Fla.).
As shown in FIG. 2A, the conjugate retains saporin activity when
tested in an in vitro protein synthesis inhibition assay. The
conjugate, as expected, is slightly less active (about two-fold)
than free SAP. This is consistent with the low level of
derivatization of SAP prior to the conjugation of (0.8 moles
SPDP/mole) and with probably steric hindrance due to the presence
of bound basic FGF. In contrast, the results obtained in the
radioreceptor assays for basic FGF (FIG. 2B) showed that the basic
FGF-SAP is equipotent to, if not slightly more active than, basic
FGF in the binding assay. Thus, it appears that the commitment of
free sulfhydryl groups in basic FGF to bridging with SAP does not
interfere with its capacity to recognize its receptor. If anything,
this reaction may be stabilizing basic FGF.
Basic FGF-SAP is a potent cytotoxic factor for BHK cells (FIG. 3).
SAP has no toxic effect on these cells even at the highest dose
tested (10.sup.-8 M) and basic FGF alone has a slight inhibitory
effect on proliferation. A mixture of basic FGF and SAP had a
slight toxicity but only at the highest concentration tested. The
ID.sub.50 (25 pM) for the cytotoxic agent compared well with the
potency of basic FGF (15 pM) in proliferation assays. Specificity
of the cytotoxic agent was examined in competition experiments in
an effort to establish that the mitotoxic activity of the conjugate
is receptor specific. BHK cells were preincubated for one hour with
various levels of basic FGF or nerve growth factor (NGF) prior to
treatment of the cells with the cytotoxic agent. As shown in FIG.
4, there is a dose-related inhibition of the cytotoxic activity in
the presence of increasing amounts of basic FGF. In contrast, a
thousand-fold excess of NGF has no effect.
EXAMPLE III
Inhibition of Angiogenesis in Rabbit Cornea
Elvax (ethylene-vinyl acetate copolymer resin, Dupont, Wilmington,
Del.) pellets were produced in the following manner. About 60 mg of
washed and dried Elvax was dissolved in 500 .mu.L of methylene
chloride. This was added to 50 .mu.g of dried basic FGF. 5 .mu.L
drops were dropped onto a slide frozen in dry ice. Pellets were
left in the freezer overnight and then dried in a desiccator.
New Zealand white rabbits were anaesthetized with Innovar Vet: 1
mL/kg. An incision was made in the cornea of the rabbit eye and a
pocket was opened with spatula or forceps. One pellet was inserted
in the pocket. Pellets were inserted in both eyes. The eye was
washed with saline, and 1 ml of gentamicin was injected
intramuscularly. The rabbit was left for five days, and
angiogenesis was observed. After five days, each left eye was
treated with 20 .mu.L of 100 ng basic FGF-SAP prepared as in
Example I in 0.25% BSA. The right eyes were treated with 20 .mu.L
of 0.25% BSA alone. The treatment was done twice daily by dropping
the solution as eye drops onto the cornea of the rabbit. The person
treating the animals was unaware of the identity of the samples.
After 10 days, the animals were evaluated for angiogenesis of the
cornea by microscopic analysis by an evaluator who did not know the
treatment regimen. Angiogenesis was judged, with +++ as being
maximal angiogenesis and -- as being no angiogenesis.
The results are provided in Table I. As can be seen, angiogenesis
in corneas treated with basic FGF-SAP was markedly reduced over
that of controls.
TABLE I ______________________________________ ANIMAL RIGHT EYE
LEFT EYE ______________________________________ 995 + - 997 +++ +
998 +++ + 999 ++ - ______________________________________
EXAMPLE IV
Effect of FGF-SAP in Dupuytren's Cell
Cells obtained from surgical removal of tissue from the hand of
adult patients diagnosed as having Dupuytren's Contracture, a
malady effecting movement of the hand, were placed in primary
culture. These cells have between 10,000 and 15,000 basic FGF
receptors per cell.
The cells were grown overnight in a 24-well tissue culture dish at
a concentration of 10,000 cells per well in HEPES-buffered DMEM
with 10% FCS. The next morning the media was removed and replaced
with media containing concentrations of basic FGF-SAP conjugate
ranging from 10.sup.-8 to 10.sup.-12 molar. Controls included wells
treated with media only, and wells treated with similar
concentrations of basic FGF alone, saporin alone, and basic FGF and
saporin together but not conjugated. The cells were returned to the
incubator for 72 hours. At the end of this incubation, the cells
were washed, removed with trypsin and counted on a Coulter cell
counter. The number of cells in the media controls was compared
with the number of cells in the treated wells (as described above).
The results of these cell killing assays are shown in FIG. 6. As
can be seen, Dupytren's cells are sensitive to basic FGF-SAP.
Similar results were obtained with three other cell samples.
Pseudomonas exotoxin (PE) is substituted for saporin in the
protocol of Example I, derivatized with SPDP and conjugated with
basic FGF. The bFGF-PE conjugate is employed as described in
Examples II, III, and IV for bFGF-SAP and similar results are
obtained.
In addition, acidic FGF is substituted for basic FGF in the Example
I protocol and mixed with the derivatized saporin; the conjugate is
purified as generally described in Example I. The aFGF-SAP
conjugate is employed as described in Examples II, III, and IV for
bFGF-SAP and similar results are obtained. The peptide
FGF-(93-120)-NH.sub.2 (amino acids 93-120 of basic FGF having 146
amino acid residues, see U.S. Pat. No. 5,132,408) is substituted
for basic FGF in the above protocol and mixed with the derivatized
saporin; the conjugate is purified as generally described in
Example I. The FGF-(93-120)-SAP conjugate is employed as described
in Examples II, III, and IV for bFGF-SAP and similar results are
obtained.
Although the invention has been described with reference to the
presently-preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
* * * * *